NPM-ALK and ALK in Lymphoma and Normal Development

在非霍奇金淋巴瘤（NHL）和炎性肌纤维母细胞瘤（IMT）肉瘤中，间变性淋巴瘤激酶（ALK）受体酪氨酸激酶（RTK）会因染色体重排而改变，从而产生组成型活性ALK融合蛋白。ALK融合表达与NHL或IMT的发生存在直接的因果关系。相反，抑制ALK活性可以在实验中逆转恶性表型。ALK的正常功能尚不完全清楚，ALK敲除小鼠没有表现出明显或组织病理学异常。ALK信号转导的研究已经确定了几种先前已知的激酶底物在介导ALK信号转导中起重要作用，但激酶正常或致癌信号转导所需的底物的完整谱尚不清楚。鉴于ALK作为定向治疗靶点的良好验证，本提案的Aim 1a旨在开发atp竞争的ALK小分子抑制剂，并确定其在临床前肿瘤模型中的特异性、毒性和有效性。为了为合理的、基于结构的ALK抑制剂设计提供指导，Aim 1b的研究将确定载脂蛋白、失活的、三磷酸化的、完全活性的ALK KD的x射线晶体结构，以及与选定的抑制剂共结晶的ALK KD的结构。被称为白细胞酪氨酸激酶（LTK）的RTK和ALK具有显著的同源性和部分重叠的表达模式，提示可能的功能冗余。为了更好地了解这些激酶在生长和发育中的功能，并评估它们同时抑制的后果（即使是高度特异性的atp竞争性ALK抑制剂也可能在药理学上发生），我们将生成缺乏ALK和Ltk的小鼠，并在Aim 1c中进行表征。最近的ALK信号研究发现了一种新的底物，ALK相互作用核蛋白3 (ANP-3)，它具有改变细胞凋亡和细胞周期转运的能力，特别是有丝分裂的进入和退出。Aim 2a的研究将试图确定ANP-3在凋亡死亡和细胞周期的正常调节中的确切作用，特别是关注m期转运，并了解ANP-3通过致癌ALK融合解除调控的机制和后果。最后，在Aim 2b中，将评估体内Anp-3失活对胚胎发育、细胞周期完整性和细胞凋亡控制的表型影响。这些研究将提高对正常ALK功能和ALK融合蛋白在恶性肿瘤中的作用机制的基本认识，同时也将确定小分子ALK抑制剂作为基础研究工具，并最终可能用于治疗应用。

The anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK) is altered by chromosomal rearrangements in non- Hodgkin's lymphoma (NHL) and the sarcoma known as inflammatory myofibroblastic tumor (IMT) to produce constitutively active ALK fusion proteins. A direct causal relationship exists between the expression of ALK fusions and the genesis of NHL or IMT. Conversely, inhibition of ALK activity reverses the malignant phenotype experimentally. The normal functions of ALK are incompletely known, and Alk knockout mice exhibit no gross or histopathologic abnormalities. Studies of ALK signal transduction have identified several previously known kinase substrates as important in mediating ALK signaling, but the complete spectrum of substrates required for normal or oncogenic signaling by the kinase is not yet clear. Given the excellent validation of ALK as a target for directed therapies, Aim 1a of this proposal seeks to develop ATP-competitive small molecule inhibitors of ALK and to determine their specificity, toxicity and efficacy in preclinical tumor models. To provide guidance for the rational, structure-based design of ALK inhibitors, the studies in Aim 1b will determine the X-ray crystallographic structure of the apo, inactive and tris-phosphorylated, fully active ALK KD, as well as the structure of the ALK KD co- crystallized with selected inhibitors. The RTK known as leukocyte tyrosine kinase (LTK) and ALK share remarkable homology and a partially overlapping expression pattern, suggesting possible functional redundancy. To better understand the function of these kinases in growth and development and assess the consequences of their simultaneous inhibition (as may occur pharmacologically even with highly specific ATP-competitive ALK inhibitors), mice that lack both Alk and Ltk will be generated and characterized in Aim 1c. Recent ALK signaling studies have identified a novel substrate, ALK-interacting nuclear protein 3 (ANP-3), that possesses the ability to alter both apoptosis and cell cycle transit, especially mitotic entry and exit. The studies in Aim 2a will seek to determine the exact role of ANP-3 in the normal regulation of apoptotic death and the cell cycle, focusing especially on M-phase transit, and to understand the mechanism and consequences of ANP-3 deregulation by oncogenic ALK fusions. Finally, in Aim 2b, the phenotypic effects of Anp-3 inactivation in vivo on embryonic development, cell cycle integrity and apoptosis control will be assessed. These studies will improve basic understanding of normal ALK functions and the mechanisms employed by ALK fusion proteins in malignancy, while also identifying small molecule ALK inhibitors for use as basic research tools and ultimately perhaps for therapeutic applications.